Confocal Microscope C2+

C o n f o c a l M i c r o s c o p e

An essential microscopy laboratory instrument…

The C2+ confocal microscope system is part of a new generation of Nikon confocal

instruments designed to be essential laboratory microscopy tools. Built on a

reputation of incredible stability and operational simplicity coupled with superior

optical technologies and high-speed image acquisition of up to 100 fps*, the C2+ is

the perfect tool for a new microscope, or as a new accessory to an existing Nikon

imaging system.

Large field-of-view imaging and three-dimensional reconstruction

Confocal image acquisition, using high-numerical aperture and high-magnification objectives, togetherwith XY stage control and advanced image stitching with Nikon NIS-Elements software, enables high-resolution images of large areas of a specimen to be produced. In addition, the microscope’s high-precision Z-axis control allows assemblage of Z stack images for three-dimensional imagereconstruction.

2 3

Confocal Microscope

Specimen: Genital tract of DrosophilamelanogasterPhoto courtesy of: Director and ProfessorMasatoshi Yamamoto, Drosophila GeneticResource Center, Kyoto Institute ofTechnology

* With 8x zoom or larger

2.3mm

Testis

Seminal Vesicle

Ejaculatory Duct

Ejaculatory Bulb

Accessory Gland

Image Stitching (Large Image)

4 5

High-efficiency scanning heads and detectors

Nikon's unprecedented optics and highly efficient optical design provide the brightest and sharpestimages, at the longest working distances.

Image QualityHigh-performance imaging software NIS-Elements offers a variety of image processingand analysis functions. It also enables data extraction from acquired images. In addition,NIS-Elements allows for intuitive operation of Nikon microscopes and other third-partyperipheral devices, such as EMCCD cameras and filter wheels, to broaden the range ofexperiments possible.

High functionality

With the convenient, small scan head size, the C2+ can be used with various typesof Nikon microscope. The C2+ employs high precision mirrors and optically superiorcircular pinholes, and separates the detectors to isolate sources of heat and noise,enabling low-noise, high-contrast and high-quality confocal imaging.The newly developed scanner driving system and Nikon’s unique image correctiontechnique allow 8 fps (512 x 512 pixels) and 100 fps (512 x 32 pixels) high-speedimaging.

Multimode capabilityVarious imaging methods, such as confocal, widefield, TIRF, photoactivation,as well as processing, analysis and presentation of acquired images, areavailable in one software package. Users can easily learn how to controldifferent imaging systems with a common interface and workflow.

High-performance opticsCFI Apochromat �S SeriesThese high-numerical aperture (NA) objectives are ideal for confocal imagingwith correction of chromatic aberrations over a wide wavelength range fromultraviolet. In particular, the LWD 40xWI lens corrects up to infrared.Transmission is increased through the use of Nikon’s exclusive Nano CrystalCoat technology.

CFI Apochromat TIRF SeriesThese objectives boast an unprecedented NA of 1.49 (using a standardcoverslip and immersion oil), the highest resolution among Nikon objectives.The temperature correction ring adjusts for image quality affected bytemperature change in the range of 23°C to 37°C.

High-definition diascopic DIC imagesThe C2+ can acquire simultaneous three-channel fluorescence or simultaneousthree-channel and diascopic DIC observation. High-quality DIC images andfluorescence images can be superimposed to aid in morphological analysis.

DIC image Overlay of DIC and fluorescence images

CFI Apochromat 40xWI �S, NA1.25 (left) CFI Apochromat LWD 40xWI �S, NA1.15 (middle) CFI Apochromat 60x Oil �S, NA1.4 (right)

CFI Apochromat TIRF 60x oil/1.49 (left)CFI Apochromat TIRF 100x oil/1.49 (right)

Scanning parameter settingsSimple GUI: Simple display offundamental image acquisition settings

Setting: Easy-to-recognize display for setting lasers,detectors, etc.

nDtime, nDXYZ: Intuitive settings for time schedule and Z series parameters, etc.

Optical Config: Multiple setting parameters (such as camera settings andchannel setup) can be extracted from acquired images and registered for reuse.

Spectral analysis GUI: Numerous functions for analysis and unmixing of acquiredspectrums are provided, while spectral profiles of general dyes and fluorescent proteinsare preprogrammed.

In addition to the conventional three-channel fluorescence detector, the C2si+ true spectral imaging confocal laserscanning microscope is equipped with a dedicated spectral detector. By switching between these detectors, accuratespectral data of fluorescence signals can be obtained. The C2si+ captures minute changes of wavelength in truecolor and even unmixes overlapping spectra. Moreover, it has the capability to acquire spectra over a 320 nm-widewavelength range in a single scan, minimizing damage to living cells.

True Spectral Imaging

Multi-anode PMTThe spectral imaging detector utilizes a lasershielding mechanism. Coupled with a wavelengthresolution independent of pinhole diameter, thismechanism successfully shuts out the reflected laserbeam. The blocking mechanism can be moved freelywith software, allowing users to block any laserwavelength, making the C2si+ compatible withvirtually any laser selection.

DEES systemHigh diffraction efficiency is achieved bymatching the polarization direction of lightentering a grating to the polarizing light beam S. Unpolarized light

Polarized beam splitter

Optical fiberThe wavelength resolution isindependent of pinhole diameter.

S1

P

S1

S2

S2Polarization rotator

Multiple gratingsWavelength resolution can be varied between2.5/5/10 nm with three gratings. Each position isprecisely controlled for high-wavelengthreproducibility.

High-quality spectral data acquisition

(Brightness)

(Channel)

2000

2500

3000

3500

4000

1 4 7 10 13 16 19 22 25 28 31

Pre-correction (Brightness)

(Channel)

2000

2500

3000

3500

4000

1 4 7 10 13 16 19 22 25 28 31

Post-correction

Multi-anode PMT sensitivity correction

Accurate, reliable spectral data: three correction techniquesThree correction techniques allow for the acquisition of accurate spectra:interchannel sensitivity correction, which adjusts offset and sensitivity of eachchannel; spectral sensitivity correction, which adjusts diffraction grating spectralefficiency and detector spectral sensitivity; and correction of spectral transmissionof optical devices in scanning heads and microscopes.

Diffraction Efficiency Enhancement System (DEES)With the DEES, unpolarized fluorescence light emitted by the specimen isseparated into two polarizing light beams P and S by a polarizing beamsplitter. P is converted by a polarization rotator into S, which has higherdiffraction efficiency than P, achieving vastly increased overall diffractionefficiency.

0

400 750

10

20

30

40

50

60

70

80

90

100

Wavelength (nm)

Dif

frac

tio

n e

ffic

ien

cy (

%) S polarizing light beam

P polarizing light beam

Characteristics of grating

High-efficiency fluorescence transmission technologyThe ends of the fluorescence fibers and detector surfaces use a proprietary anti-reflective coating to reduce signal loss to a minimum, achieving high opticaltransmission.

Effortless fluorescence unmixingFluorescence labels with closely overlapping spectra can be unmixed cleanlywith no crosstalk. Even without a given reference spectrum, simply specifyinga Region of Interest (ROI) within the image and clicking the Simple Unmixingbutton allows separation of fluorescence spectra. The C2si+ contains a built-in

Intensity

Wavelength

f f

λ

1 1

n

P nS 2 2P nS

The spectrum obtained by actual measurement is a mix of spectral elements with a certain proportion. An imagingalgorithm is used to compare the spectra of each pixel with reference curves for each spectral element. Each fluorescentprobe in the specimen is displayed in a unique color in the final unmixed image.

Reference wave pattern (S) is selected from the following three depending on the experiment.1 Spectrum obtained by actual measurement of the zone with less crosstalk in the captured image2 Data obtained by another actual measurement using only one probe3 Spectral data provided by probe maker

What is spectral unmixing?

fn=Sn＊Pfn = Wave pattern of spectrum obtained by actual measurement

Sn= Wave pattern of individual reference spectrum

P = Ratio of elements for each wave pattern

Fluorescenceunmixing

Specimen: HeLa cell in which GFP (Tubulin) and YFP (Golgi) are expressed. Spectral image captured with a 488 nm laser (left). After fluorescence unmixing,GFP is indicated in green and YFP is indicated in red (right). The graph (left) shows the spectral curve in the ROI.Specimen courtesy of Dr. Sheng-Chung Lee, Dr. Han-Yi E. Chou, National Taiwan University College of Medicine, Institute of Molecular Medicine

database of given spectral data provided by manufacturers of fluorescencedyes that can be specified as reference spectra for fluorescence unmixing.Users may also add spectral information for new labels to the database.

6 7

Confirmation of GFP expressionIn conventional confocal observation, fluorescence is visualized as fluorescence intensityin a certain wavelength range. The spectral detector allows the confirmation of detailedwavelength characteristics of the fluorescence. The C2si+’ spectral detector enables theslight color differences to be confirmed as wavelengths through sensitivity correction.

Unmixing auto-fluorescence of multi-stained samplesFluorescence unmixing makes it possible not only to separate closely overlappingfluorescence spectra such as CFP and YFP but also to eliminate auto-fluorescence of cells,which until now was difficult.

Specimen: actin of HeLa cell that has RFP expressed in the nucleus is stained with Rhodamine. Spectral image in the 550-630 nm wavelength range captured at 2.5 nm wavelength resolutions with 543 nm laser exposure (left). RFP indicated inred and Rhodamine indicated in green (right) in the image after fluorescence unmixing.Specimen courtesy of Dr. Yoshihiro Yoneda and Dr. Takuya Saiwaki, Faculty of Medicine, Osaka University

Specimen: HeLa cell in which nucleus is labeled with CFP, actin-related protein (Fascin) labeled with GFP, Golgi body labeledwith YFP, and mitochondria labeled with DsRed. Spectral image captured with 408 nm and 488 nm laser exposure (left). The fluorescence spectra of the captured image are unmixed using reference spectra (right).Specimen courtesy of Dr. Kaoru Kato and Dr. Masamitsu Kanada, Neuroscience Research Institute, The National Institute ofAdvanced Industrial Science and Technology (AIST)

Specimen: Zebrafish egg stained with cadherin-GFP and DAPI. Spectral image captured with 408 nm and 488 nm laserexposures (left). After unmixing using reference spectra for auto-fluorescence (ROI1), GFP and DAPI, the auto-fluorescence inthe image is eliminated (right).Specimen courtesy of Dr. Tohru Murakami, Neuromuscular and Developmental Anatomy, Gunma University Graduate Schoolof Medicine

Specimen: Arabidopsis proteoglycan and fused protein of GFP. Spectral image captured with 488 nm laser exposure (left).Once the image is unmixed using reference spectra for auto-fluorescence (ROI1) and GFP, GFP is indicated in green andauto-fluorescence is indicated in red (right).Specimen courtesy of Assistant Prof. Toshihisa Kotake, Laboratory of Developmental Biology, Department of Life Science,Graduate School of Science and Engineering, Saitama University

Unmixing red fluorochromesRed fluorochromes, which had previously posed a challenge, are now simple to unmix.

Spectra for ROI 1 and 2 corresponding to the image on the rightRhodamine’s fluorescence spectral peak is at approximately 579 nm,while that for RFP is approximately 600 nm. RFP’s fluorescence isweaker than Rhodamine’s, but their spectra are cleanly unmixed.

Unmixing of multiple fluorescenceBecause wavelength resolution and range are freely selectable, scanning of a fluorescenceprotein with a wide wavelength range from blue to red such as CFP/GFP/YFP/Ds Red ispossible at one time. Reference data allows unmixing and display of each color.

Fluorescenceunmixing

Fluorescenceunmixing

Fluorescenceunmixing

Fluorescenceunmixing

The correspondence of the spectral curve (blue) of ROI2 inthe image and the reference curve (green) of eGFP provesthat GFP is expressed in ROI2

xy

xz

yz

xy

xz

yz

FRET (Fluorescence Resonance Energy Transfer) analysis using true spectralimaging allows three-dimensional analysis with high signal-to-noise (S/N)ratio and high-spatial resolution as well as easy determination of FRET byreal-time detection of spectral changes derived by FRET.

Spectral image in the 460-620 nm range captured at 5 nm wavelengthresolution using a spectral detector enables observation of fluorescencewavelength changes.

True color image and spectral analysis of CFP and YFP. Spectral curve in ROI. Leftpeak indicates CFP and right peak indicates YFP respectively. After ATP stimulation,peak of CFP drops and peak of YFP rises due to FRET.

Before ATP stimulationTrue color image

Spectral analysis

8 sec after ATP stimulation

Spectral FRET analysis is possible by unmixing using reference data of CFP and YFP.Two-dimensional change (FRET) of intracellular Ca2+ concentration is easily determinedfrom spectral data without acceptor bleaching.

FRET image after spectral unmixing. CFP is indicated in blue and YPF indicated in green.

Time-lapse changes (T) and spectra (λ) in three-dimensional space (XYZ) canbe analyzed.

Before ATP stimulation 8 sec after ATP stimulation

Also, even when spectra of donor and acceptor are overlapped like CFP andYFP, unmixing using reference data enables detection of detailed intensitychanges and ratio analysis of fluorescence signals (YFP/CFP) without bleedthrough.

Acquisition of spectral image (XYTλ) Fluorescence unmixing

Five-dimensional analysis (XYZTλ)

True spectral FRET analysis

8 9

6 7

X position (pixel) X position (pixel)

L1L2L3L4

PC

Software

Diascopic Detector Unit*6

AOM Unit

Laser unit

Scanner set

Microscope

Detector unit

1st DM 1st DM

Filter Cube

Spectral Detector

C-LU3EX 3-laser Module EX

Standard Epi-fl Detector (3-PMT)

LU-LR 4-laser Power Source Rack

C2 Scanning Head

C2si Scanning Head

AZ100*5FN1*2 Ni-E/Ni-U*3

Controller

LU4A 4-laser Module A

Ring Adapter SRing Adapter L/S/SS Ring Adapter L/S/SS

*1 Not required when using a Stage-up Kit.*2 C-TT-C Trinocular Tube, C1 Y-TT Trinocular Tube or NI-TT Quadrocular Tilting tube can be used.*3 With Ni-U, NI-TT Quadrocular Tilting tube or C-TT-C Trinocular Tube can be used. With Ni-E, NI-TT-E Motorized Quadrocular Tilting tube, NI-TT Quadrocular Tilting tube or C-TT-C Trinocular Tube (for Ni-E focusing stage only) can be used.*4 Required when using NI-TT Quadrocular Tilting tube or NI-TT-E Motorized Quadrocular Tilting tube.*5 Use AZ-TE100LS Ergonomic Trinocular Tube 100 LS and AZ100 Stage Cover.*6 Dedicated adapter is required depending on microscope model.

C2-NI-TT Quadrocular Tilting Tube A*4C1-TI TI Mounting Adapter

Ring Adapter S*1

Z-focus ModuleZ-focus Module (for Ti-U)

Z-focus Module(for Ni-U) Z-focus Module

Ti-E/U

Ｆ．ＳＴＯＰ Ａ．ＳＴＯＰ

ＥＸ．ＡＤＪ．

10 11

System diagramThe C2+ can be coupled with upright, inverted, physio-logical, and macro imaging microscopes and has options forcombinations with various high-quality research experimentsystems. All can be controlled with NIS-Elements software.

Flexibility

Optional TIRF laser illumination module and a photoactivation module canbe integrated to enable both imaging of single molecules with an extremelyhigh S/N ratio, and imaging of the fluorescence characteristic changes ofphotoactivated and photo-convertible fluorescent protein.

TIRF/Photoactivation-C2+ Multimode imaging system

With a high-definition large field of view, specimens larger than 1cm can beacquired with an unprecedentedly high S/N ratio. The AZ-C2+ allows forimaging of whole-mount specimens, such as embryos, in a single acquisition,up to 4000x4000 pixel resolution, and it can also acquire 32-channel spectraldata with the C2si+. It offers a combination of low and high magnificationobjective lenses, optical zoom and a confocal scanning zoom function,enabling continuous imaging from macro to micro.

AZ-C2+ Macro confocal microscope system

TT2 ES cells Anti-Nanog antibody (Cy3), anti-Oct3/4 antibody (Alexa488)and DAPI localized in cell nuclei Photographed with the cooperation of: Hiroshi Kiyonari,Laboratory for Animal Resources and Genetic Engineering,RIKEN Center for Developmental Biology

Photo courtesy of: Director and Professor Masatoshi Yamamoto,Drosophila Genetic Resource Center, Kyoto Institute of Technology

EnPrinted in Japan (1112-00)T Code No. 2CE-SCHH-3This brochure is printed on recycled paper made from 40% used material.

Specifications and equipment are subject to change without any notice or obligationon the part of the manufacturer. December 2011 ©2010-11 NIKON CORPORATION

Monitor images are simulated.Some sample images in this brochure were captured using the C1 confocal microscope system.Company names and product names appearing in this brochure are their registered trademarks or trademarks.N.B. Export of the products* in this brochure is controlled under the Japanese Foreign Exchange and Foreign Trade Law.Appropriate export procedure shall be required in case of export from Japan.*Products: Hardware and its technical information (including software)

WARNINGTO ENSURE CORRECT USAGE, READ THE CORRESPONDINGMANUALS CAREFULLY BEFORE USING YOUR EQUIPMENT.

NIKON CORPORATIONShin-Yurakucho Bldg., 12-1, Yurakucho 1-chome, Chiyoda-ku, Tokyo 100-8331, Japan phone: +81-3-3216-2375 fax: +81-3-3216-2385http://www.nikon.com/instruments/

Compatible laser*1 405 nm/440 nm, 488 nm/Ar laser (457 nm, 477 nm, 488 nm, 514 nm), 543 nm/561 nm/594 nm, 633 nm/638 nm/640 nm

Laser unit C-LU3EX 3-laser Module EX (AOM or manual modulation), LU4A 4-laser Module A (AOTF modulation)

Standard detector Wavelength: 400-750 nm, Detector: 3PMT, Filter cube: 2 filter cubes

Diascopic detector (option) Wavelength: 400-700 nm, Detector: 1 PMT

Scanning head (galvano) With Standard detector: Pixel size: 2048 x 2048 Scanning speed: Standard mode: 2 fps (512 x 512 pixels, bi-direction), 17 fps (512 x 32 pixels, bi-direction), Zoom: 1-1000x Fast mode: 8 fps (512 x 512 pixels, bi-direction), 100 fps (512 x 32 pixels, bi-direction)*2, Zoom: 8-1000x

With spectral detector: Pixel size: max. 1024 x 1024 pixels Scanning speed: 0.5 fps (512 x 512 pixels, single direction), max. 6 fps (64 x 64 pixels, single direction)

Scanning mode X-Y, XY rotation, zoom, ROI, XYZ, time lapse, X-Z, stimulation, multipoint, image stitching (large image)

Pinhole Circular shape, 6 size

Spectral detector Number of channels: 32 channels, Wavelength: 400-750 nm, (with galvano scanner) Wavelength resolution: 2.5 nm, 5 nm, 10 nm, wavelength range variable in 0.25 nm steps, (option) Unmixing: High-speed unmixing, precision unmixing

FOV Square inscribed in a ø18 mm circle

Image bit depth 12 bits

Compatible microscopes ECLIPSE Ti-E/Ti-U inverted microscope, ECLIPSE Ni-E (focusing nosepiece type/stage focusing type)/Ni-U upright microscope, ECLIPSE FN1 fixed stage microscope, AZ100 multi-purpose zoom microscope

Z step Ti-E: 0.025 µm, FN1 stepping motor: 0.05 µm, Ni-E: 0.025 µm

NIS-Elements C software Display/image processing/analysis 2D/3D/4D analysis, time-lapse analysis, 3D volume rendering/orthogonal, spatial filters, image stitching, multipoint time-lapse, spectral unmixing, real-time unmixing, virtual filters, deconvolution, AVI image file output

Application: FRAP, FLIP, FRET, photoactivation, colocalization, three-dimensional time-lapse imaging, multipoint time-lapse imaging

Control Computer OS: Microsoft Windows 7 Professional 64 bit SP1 (Japanese/English), CPU: Intel Xeon X3565 (3.20 GHz/8 MB/1066 MHz/Quad Core) or higher, Memory: 4 GB, Hard disk: 146 GB SATA 3 Gb/s (7200 rpm), Data transfer: LAN, Network interface: Gigabit Ethernet, Monitor: 1600 x 1200 or higher resolution, dual monitor configuration is recommended

Specifications

C2+

Recommended layout

380 700

650 (7

85)

405

225 510 W

1450

700

(2400 or 2900)

Controller

レーザユニット Scanning Head

Spectral Detector

Standard Epi-fl Detector

4-laser Unit

W=1500mm (two 19-inch monitors)W=1000mm (24-inch monitor)

Note) Computer table size is for reference only.

L1L2L3L4

L1 POWERL2L3L4

852

unit: mm

NIKON INSTRUMENTS INC.1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A.phone: +1-631-547-8500; +1-800-52-NIKON (within the U.S.A. only)fax: +1-631-547-0306http://www.nikoninstruments.com/

NIKON INSTRUMENTS EUROPE B.V.Laan van Kronenburg 2, 1183 AS Amstelveen, The Netherlandsphone: +31-20-44-96-300 fax: +31-20-44-96-298http://www.nikoninstruments.eu/

NIKON INSTRUMENTS (SHANGHAI) CO., LTD.CHINA phone: +86-21-6841-2050 fax: +86-21-6841-2060(Beijing branch) phone: +86-10-5831-2028 fax: +86-10-5831-2026(Guangzhou branch) phone: +86-20-3882-0552 fax: +86-20-3882-0580

NIKON SINGAPORE PTE LTDSINGAPORE phone: +65-6559-3618 fax: +65-6559-3668

NIKON MALAYSIA SDN. BHD.MALAYSIA phone: +60-3-7809-3688 fax: +60-3-7809-3633

NIKON INSTRUMENTS KOREA CO., LTD.KOREA phone: +82-2-2186-8400 fax: +82-2-555-4415NIKON CANADA INC.CANADA phone: +1-905-602-9676 fax: +1-905-602-9953NIKON FRANCE S.A.S.FRANCE phone: +33-1-4516-45-16 fax: +33-1-4516-45-55

NIKON GMBHGERMANY phone: +49-211-941-42-20 fax: +49-211-941-43-22

NIKON INSTRUMENTS S.p.A.ITALY phone: +39-055-300-96-01 fax: +39-055-30-09-93

NIKON AGSWITZERLAND phone: +41-43-277-28-67 fax: +41-43-277-28-61

NIKON UK LTD. UNITED KINGDOM phone: +44-208-247-1717 fax: +44-208-541-4584

NIKON GMBH AUSTRIA AUSTRIA phone: +43-1-972-6111-00 fax: +43-1-972-6111-40

NIKON BELUXBELGIUM phone: +32-2-705-56-65 fax: +32-2-726-66-45

*1 Compatible lasers and usable wavelengths differ depending on the laser unit in use.*2 The described frame rate is NOT available with Rotation, CROP, ROI, Spectral imaging and Stimulation.